Technical methods
486
Technical methods Simple method for the cytological examination of cerebrospinal fluid Department ofPediatrics, St. Louis University School of Medicine and Cardinal Glennon Memorial Hospital for Children, St. Louis, Missouri, USA
J.-Y. CHU, P. FREELING, AND S. WASSILAK
The cytological examination of cerebrospinal fluid (CSF) is important in the diagnosis of central nervous system leukaemia and other diseases. However, it is not easy to prepare a good smear because the cells are easily lysed after a short period and also are easily damaged by centrifugation, fixation, and concentration (S6rnas, 1967). The sedimentation method of S6rnas appears to be a much better method for both cell recovery and morphology (Sornas, 1967; Aronson et al., 1974). We have deAddress for correspondence and reprints: J.-Y. Chu, MD, Cardinal Glennon Memorial Hospital for Children, 1465 S. Grand Blvd, St. Louis, Missouri 63104, USA.
Received for publication 29 November 1976
Fig. 1
Fig. 2
veloped a simpler and more practical sedimentation method which is reported below. Material and methods A thick circle, about 1P5 cm in diameter, was made on a clean fat-free glass slide with a laboratory wax pencil. Fresh CSF was then applied dropwise within the circle; 0-2-0-3 ml of CSF was retained in the circle without difficulty. The slide was then placed in a Petri dish with water at the bottom to provide saturated humidity. After sedimentation for 15-30 minutes the fluid was absorbed with the tip of a piece of blotting paper and dried with an air jet. Slides were then stained with Wright stain and mounted. The area within the circle was searched with a low-power lens to locate the suspicious cells which were then examined under higher power. Annoying debris from wax at the edge of the circle can be reduced by briefly flaming the slide beforehand, and wax will adhere to the glass better. The circle can also be easily made by Permount (histological mounting medium, Fisher Scientific Co, Fair Lawn, New Jersey). We apply Permount to a clean cut edge of rubber tube and then compress the edge of the tubing to the glass.
Fig.3
Fig. 1 Cytocentrifuge preparation of CSFfrom a patient with Chediak-Higashi's syndrome showing spreading of cytoplasm and an obscure granule. Fig. 2 Sedimentation preparation of CSF from a patient with Chediak-Higashi's syndrome showing better morphology and a giant granule. Fig. 3 Leukaemic cell in sedimentation preparation containing only a few cells per ul of CSF. 486
487
Technical methods Results and discussion
less than 30 minutes. The cells, of course, are less concentrated in our method than in that of Sornas. However, the same amount of CSF will be adequate to make more slides. By using low-power lens for scanning the suspicious cells and oil lens for detailed morphology, we probably examine as many cells as in Sornas' method. In a few patientswhoseCSF contained few cells per microlitre the smear revealed a few definitely leukaemic cells (Fig. 3).
After evaluation and comparison of many different methods, Sornas developed and recommended the sedimentation method for cytology of CSF (S6rnas, 1967; Aronson et al., 1974). The cytocentrifuge method has also been recommended recently by a few investigators (Drewinko et al., 1973; Evans et al., 1974). We have found much cell distortion with the cytocentrifuge method and very poor yields, as has been reported by others (Addiego and Woodruff, 1973). The distortion of cell morphology, especially References the spreading of the cytoplasm, makes interpretation difficult (Fig. 1). Reduced distortion of cell Addiego J. E. and Woodruff, K. (1973). Cytocentrifugation in central nervous system leukemia. J. Pediat., 82, morphology was well demonstrated in CSF from a 891-892. patient with Chediak-Higashi's syndrome. Giant A. S., Garwicz, S., and Srnais, R. (1974). granules in the cytoplasm of lymphocytes were Aronson, Cytology of the cerebrospinal fluid in children with seldom seen in cytocentrifuge preparations but were acute lymphoblastic leukemia. J. Pediat., 85, 222-224. well demonstrated in the preparations by our sedi- Castleberry, R. P., Moreno, H., and Wallace, L. S. (1975). mentation method (Fig. 2). Cytologic analysis of cerebrospinal fluid. J. Pediat., 86, The method described here is also simpler and 989-990. more practical. It is difficult to scrape off completely Drewinko, B., Sullivan, M. P., and Martin, T. (1973). Use of the cytocentrifuge in the diagnosis of meningeal the petroleum jelly in the method of Sornas (1967), leukemia. Cancer (Philad.), 31, 1331-1336. and this method has also been criticised as timeconsuming (Castleberry et al., 1975). Our method Evans, D. I. K., O'Rourke, C., and Jones, P. M. (1974). The cerebrospinal fluid inacuteleukaemiaofchildhood: not only eliminates the procedure of applying and studies with the cytocentrifuge. J. clin. Path., 27, 226scraping off the petroleum jelly but also shortens 230. the time considerably. Since the height of fluid within Sornas, R. (1967). A new method for the cytological the circle was no more than 3 mm, the time required examination of the spinal fluid. J. Neurol. Neurosurg. for cells to settle at the rate of 0-1 mm/min will be Psychiat., 30, 568-577.
A new method of quality control for the Coulter Model S Counter D. R. PRANGNELL AND P. H. JOHNSON Central Pathology Laboratory, Haematology Department, Hartshill Road, Hartshill, Stoke-on-Trent ST4 7PX
Conventional methods of quality control depend on the use of standards for machine calibration and the use of control samples to detect drift. The methods applied to haematology require the use of expensive whole blood reference preparations. Control samples, although they can be prepared daily or slightly less frequently (Hamilton and Davidson, 1973), must be interposed at frequent intervals between test samples. The control value can be affected by carry-over from the previous specimen (Brittin Received for publication 8 December 1976
et al., 1969) and therefore must be used twice and the second value taken as the more accurate. Control specimens may thus occupy a significant proportion of machine time. The use of a method independent of whole blood standards and controls offers a considerable saving in expense and time. The method described depends on the stability of the modal values of the population passing through the laboratory. The mode is chosen because-unlike the mean value (Cavill, 1971)-it is not affected by moderate numbers of highly abnormal results which inevitably form an unrepresentatively large proportion of the workload.
Method PREPARATION OF THE MACHINE
Recalibration of the machine is rarely necessary provided that routine cleaning is carried out. Overnight the counting orifices and diluting chambers are
486
Technical methods Simple method for the cytological examination of cerebrospinal fluid Department ofPediatrics, St. Louis University School of Medicine and Cardinal Glennon Memorial Hospital for Children, St. Louis, Missouri, USA
J.-Y. CHU, P. FREELING, AND S. WASSILAK
The cytological examination of cerebrospinal fluid (CSF) is important in the diagnosis of central nervous system leukaemia and other diseases. However, it is not easy to prepare a good smear because the cells are easily lysed after a short period and also are easily damaged by centrifugation, fixation, and concentration (S6rnas, 1967). The sedimentation method of S6rnas appears to be a much better method for both cell recovery and morphology (Sornas, 1967; Aronson et al., 1974). We have deAddress for correspondence and reprints: J.-Y. Chu, MD, Cardinal Glennon Memorial Hospital for Children, 1465 S. Grand Blvd, St. Louis, Missouri 63104, USA.
Received for publication 29 November 1976
Fig. 1
Fig. 2
veloped a simpler and more practical sedimentation method which is reported below. Material and methods A thick circle, about 1P5 cm in diameter, was made on a clean fat-free glass slide with a laboratory wax pencil. Fresh CSF was then applied dropwise within the circle; 0-2-0-3 ml of CSF was retained in the circle without difficulty. The slide was then placed in a Petri dish with water at the bottom to provide saturated humidity. After sedimentation for 15-30 minutes the fluid was absorbed with the tip of a piece of blotting paper and dried with an air jet. Slides were then stained with Wright stain and mounted. The area within the circle was searched with a low-power lens to locate the suspicious cells which were then examined under higher power. Annoying debris from wax at the edge of the circle can be reduced by briefly flaming the slide beforehand, and wax will adhere to the glass better. The circle can also be easily made by Permount (histological mounting medium, Fisher Scientific Co, Fair Lawn, New Jersey). We apply Permount to a clean cut edge of rubber tube and then compress the edge of the tubing to the glass.
Fig.3
Fig. 1 Cytocentrifuge preparation of CSFfrom a patient with Chediak-Higashi's syndrome showing spreading of cytoplasm and an obscure granule. Fig. 2 Sedimentation preparation of CSF from a patient with Chediak-Higashi's syndrome showing better morphology and a giant granule. Fig. 3 Leukaemic cell in sedimentation preparation containing only a few cells per ul of CSF. 486
487
Technical methods Results and discussion
less than 30 minutes. The cells, of course, are less concentrated in our method than in that of Sornas. However, the same amount of CSF will be adequate to make more slides. By using low-power lens for scanning the suspicious cells and oil lens for detailed morphology, we probably examine as many cells as in Sornas' method. In a few patientswhoseCSF contained few cells per microlitre the smear revealed a few definitely leukaemic cells (Fig. 3).
After evaluation and comparison of many different methods, Sornas developed and recommended the sedimentation method for cytology of CSF (S6rnas, 1967; Aronson et al., 1974). The cytocentrifuge method has also been recommended recently by a few investigators (Drewinko et al., 1973; Evans et al., 1974). We have found much cell distortion with the cytocentrifuge method and very poor yields, as has been reported by others (Addiego and Woodruff, 1973). The distortion of cell morphology, especially References the spreading of the cytoplasm, makes interpretation difficult (Fig. 1). Reduced distortion of cell Addiego J. E. and Woodruff, K. (1973). Cytocentrifugation in central nervous system leukemia. J. Pediat., 82, morphology was well demonstrated in CSF from a 891-892. patient with Chediak-Higashi's syndrome. Giant A. S., Garwicz, S., and Srnais, R. (1974). granules in the cytoplasm of lymphocytes were Aronson, Cytology of the cerebrospinal fluid in children with seldom seen in cytocentrifuge preparations but were acute lymphoblastic leukemia. J. Pediat., 85, 222-224. well demonstrated in the preparations by our sedi- Castleberry, R. P., Moreno, H., and Wallace, L. S. (1975). mentation method (Fig. 2). Cytologic analysis of cerebrospinal fluid. J. Pediat., 86, The method described here is also simpler and 989-990. more practical. It is difficult to scrape off completely Drewinko, B., Sullivan, M. P., and Martin, T. (1973). Use of the cytocentrifuge in the diagnosis of meningeal the petroleum jelly in the method of Sornas (1967), leukemia. Cancer (Philad.), 31, 1331-1336. and this method has also been criticised as timeconsuming (Castleberry et al., 1975). Our method Evans, D. I. K., O'Rourke, C., and Jones, P. M. (1974). The cerebrospinal fluid inacuteleukaemiaofchildhood: not only eliminates the procedure of applying and studies with the cytocentrifuge. J. clin. Path., 27, 226scraping off the petroleum jelly but also shortens 230. the time considerably. Since the height of fluid within Sornas, R. (1967). A new method for the cytological the circle was no more than 3 mm, the time required examination of the spinal fluid. J. Neurol. Neurosurg. for cells to settle at the rate of 0-1 mm/min will be Psychiat., 30, 568-577.
A new method of quality control for the Coulter Model S Counter D. R. PRANGNELL AND P. H. JOHNSON Central Pathology Laboratory, Haematology Department, Hartshill Road, Hartshill, Stoke-on-Trent ST4 7PX
Conventional methods of quality control depend on the use of standards for machine calibration and the use of control samples to detect drift. The methods applied to haematology require the use of expensive whole blood reference preparations. Control samples, although they can be prepared daily or slightly less frequently (Hamilton and Davidson, 1973), must be interposed at frequent intervals between test samples. The control value can be affected by carry-over from the previous specimen (Brittin Received for publication 8 December 1976
et al., 1969) and therefore must be used twice and the second value taken as the more accurate. Control specimens may thus occupy a significant proportion of machine time. The use of a method independent of whole blood standards and controls offers a considerable saving in expense and time. The method described depends on the stability of the modal values of the population passing through the laboratory. The mode is chosen because-unlike the mean value (Cavill, 1971)-it is not affected by moderate numbers of highly abnormal results which inevitably form an unrepresentatively large proportion of the workload.
Method PREPARATION OF THE MACHINE
Recalibration of the machine is rarely necessary provided that routine cleaning is carried out. Overnight the counting orifices and diluting chambers are
